Essential oil composition of Eucalyptus microtheca and Eucalyptus viminalis.

Objective: Eucalyptus (Fam. Myrtaceae) is a medicinal plant and various Eucalyptus species possess potent pharmacological actions against diabetes, hepatotoxicity, and inflammation. This study aims to investigate essential oil composition from leaves and flowers of E. microtheca and E. viminalis leaves growing in the Southeast of Iran. Materials and Methods: The aerial parts of these plants were collected from Zahedan, Sistan and Baluchestan province, Iran in 2013. After drying the plant materials in the shade, the chemical composition of the essential oils was obtained by hydro-distillation method using a Clevenger-type apparatus and analyzed by GC/MS. Results: In the essential oil of E. microtheca leaves, 101 compounds representing 100%, were identified. Among them, α-phellandrene (16.487%), aromadendrene (12.773%), α-pinene (6.752%), globulol (5.997%), ledene (5.665%), P-cymen (5.251%), and β-pinene (5.006%) were the major constituents. In the oil of E. microtheca flowers, 88 compounds representing 100%, were identified in which α-pinene (16.246%), O-cymen (13.522%), β-pinene (11.082%), aromadendrene (7.444%), α-phellandrene (7.006%), globulol (5.419%), and 9-octadecenamide (5.414%) were the major components. Sixty six compounds representing 100% were identified in the oil of E. viminalis leaves. The major compounds were 1, 8-cineole (57.757%), α-pinene (13.379%), limonene (5.443%), and globulol (3.054%). Conclusion: The results showed the essential oils from the aerial parts of Eucalyptus species are a cheap source for the commercial isolation of α-phellandrene, α-pinene, and 1, 8-cineole compounds to be used in medicinal and food products. Furthermore, these plants could be an alternative source of insecticide agents.


Introduction
Plants and their derivatives such as essential oils have long been used as food flavoring, beverages, and antimicrobial agents (Ghasemi et al., 2005). Nowadays, developing countries pay more attention to herbal medicines due to the noxious side effects of synthetic medicines on patients. In addition, the application of natural antioxidants in food factories has attracted a growing interest (Asghari and Mazaheritehrani, 2010) to minimize such oxidative damages in human body. Therefore, research works concerning essential oils as potential antioxidants for treatment of human diseases and free radical-related disorders are important. Concomitantly, public attention to natural antioxidants has been increased during the last years, and it is necessary to find natural sources of antioxidants that could replace synthetic antioxidants or at least reduce their use as food additives. For these reasons, numerous researches have been conducted in the extraction field of biologically active compounds from the herbs (Shahidi, 2000). Eucalyptus (Fam. Myrtaceae) is a genus of evergreen aromatic flowering trees, which has over 600 species (Jahan et al., 2011;Nagpal et al., 2010). It is indigenous in Australia and its Northern islands (Mozaffarian, 1996). Because of their economic value, various species of Eucalyptus are cultivated in sub-tropical and warm temperate regions (Sastri, 2002). Some of the Eucalyptus species are used for feverish conditions (malaria, typhoid, and cholera) and skin problems such as burns, ulcers, and wounds (Reynolds and Prasad, 1982). Eucalyptus species contain volatile oils that are most plentiful in the plant leaves (Pearson, 1993). Anticancer, antifungal, anti-inflammatory (Sadlon and Lamson, 2010), and antioxidant properties (Grassmann et al., 2000) have been attributed to the leaf extracts of this plant.
For this reason, the importance of these plants as an herbal medicine, the aim of the present study was to investigate the chemical composition of the essential oil from leaves and flowers of Eucalyptus microtheca and E. viminalis leaves from Zahedan (with latitude of 29° 29ˊ N and longitude of 60° 51ˊ E and 1352 m above sea level in summer of 2013) in Sistan and Baluchestan province, Iran as an important geographical zone for medicinal plants.

Plant materials
Eucalyptus microtheca and E. viminalis were collected in June, 2013 from Zahedan in Sistan and Baluchestan province (GPS coordinates: 60.8628,29.4964), Iran during the flowering stage. The taxonomic identification of each plant was confirmed by Professor V. Mozaffarian, Research Institute of Forests and Ragelands, Tehran, Iran. The voucher specimens were deposited in the national herbarium of Iran (TARI). Collected plant materials were separated with a meticulous care and dried in the shade to avoid extra damaging and minimizing crosscontamination of the plant leaves.

Isolation of the essential oil
The leaves and flowers of E. microtheca and E. viminalis leaves were dried and milled into a fine powder. The volatile oils were isolated by hydrodistillation method using a Clevenger-type apparatus. For the extraction, 50 g of the cleaned, air-dried and powdered of leave samples of E. microtheca and E. viminalis were hydro-distilled with 500 mL water in a Clevenger-type apparatus for 4 h. Moreover, 30 g of the E. microtheca flower samples were hydro-distilled with 300 mL water for 4 h. The oils were dried over anhydrous Na 2 SO 4 (Merck), stored in a dark glass bottle and kept at -8 °C until analysis.

Essential oil analysis
The essential oils were analyzed on an Agilent 6890 gas chromatograph interfaced to an Agilent 5973 N mass selective detector (Agilent Technologies, Palo Alto, USA). A fused silica capillary column (30 m length × 0.025 mm internal diameter × 0.25 μm film thickness; HP-1; silica capillary column, Agilent Technologies) was used. The data were acquired under the following conditions: The oven temperature increased from 40 °C to 250 °C at a rate of 3 °C/min.
The temperatures of injector and detector also were 250 °C and 230 °C, respectively. The carrier gas was helium (99.999%) with a flow rate of 1 ml/min and the split ratio was 50 ml/min. For GC-MS detection, an electron ionization system with ionization energy of 70 eV was used. The retention indices were calculated for all volatile constituents using retention time of n-alkanes (C 8 -C 22 ) which were injected at the same chromatographic conditions. The components were identified by comparing retention indices with those of standards. The results were also confirmed by comparing their mass spectra with the published mass spectra or Wiley library.

Results
The oils were isolated by hydrodistillation and analyzed by capillary gas chromatography, using flame ionization and mass spectrometric detection. The obtained results of the identified compounds in the essential oil of leaves and flowers of E. microtheca and E. viminalis leaves with their percentage, retention index (RI), and retention time (t R ) are shown in Tables 1, 2, and 3, respectively.

Discussion
The comparison of results showed that there are some differences and similarities between the oil compositions of these Eucalyptus species. These results are shown in Table 4. The percentages of sesquiterpene and monoterpene compounds were similar in E. microtheca leave oils, but the percentages of these components were less than those of E. viminalis leave and E. microtheca flower oil.
Studies have revealed that monoterpenes have insecticidal activities against the stored-product insects (Rajendran and Sriranjini, 2008;Papachristos et al., 2004). Our study showed that the major monoterpene compounds were in E. viminalis leave and E. microtheca flower oil. These compounds consist of 1, 8-cineole, αpinene, and β-pinene which have been shown to have insecticidal effects against some major insects that infect the stored crops (Rajendran and Sriranjini, 2008). Therefore, the essential oil of E. viminalis leaves and E. microtheca flowers from Zahedan, Iran could be a valuable alternative to chemical control strategies which have undesirable effects such as environmental pollution and direct toxicity to people. As it is evident from Table 3, the main component of the essential oils of E. viminalis leaves was 1, 8-cineole (57.757%), but it was not identified in E. microtheca leaf and flower oils. 1, 8cineole, which is a terpenoid oxide present in many plant essential oils, displays antimicrobial, anti-inflammatory, and antinociceptive effects (Juergens et al., 2003;Santos and Rao, 2000).
The percentage of α-pinene in the oil of E. microtheca flowers and E. viminalis leaves was 16.246% and 13.379%, respectively, while in E. microtheca leave oil it was less than 10%. Results indicated that some of E. microtheca leaf oil compounds such as α-phellandrene (16.487%) and aromadendrene (12.773%) were higher compared with E. microtheca flower and E. viminalis leave oils. The oil be related to the type of the plant, the plant parts (aerial or flower and leaf parts), the geographical regions of the plant growing places, and also the ecological conditions of the plant. In addition, α-pinene compound, which appeared as the major constituent in the oil of E. sargentii (20.95%), E. kruseana (17.70%), E. viminalis (13.379%), E. loxophleba (13.70%), E. dealbata (13.0%), and E. microtheca from Kashan (10.70%) and Semnan (9.47%), were present in low concentration in E. microtheca leaf oils (6.752%) from Zahedan. The amount of Pcymene compound in the oil of E. microtheca leave from Kashan (12.40%) also was much higher than that of E. gomphocephala (10.62%), E. largiflorens (10.60%), and E. microtheca (5.21%) from Zahedan.
In general, great quantitative and qualitative variations in volatile composition of E. viminalis and E. microtheca were seen between this and other studies. These variations may be due to the influence of geographical differences, environmental and growing conditions, physiological and biochemical states of plants, genetic factors, and different extraction and analytical procedures (Kokkini et al., 2004;Hassanpouraghdam et al., 2011).
It can be concluded that the oils of these two Eucalyptus species are good sources of natural antioxidants to be used in medicinal and food products to promote human health and prevent diseases, which should be investigated in further studies. In addition, regarding environmental problem and human health, these plants could be an alternative source of insecticide agents because many of their components have little or no harmful effects on humans and environment.